Abstract
Betalains replace anthocyanins as color pigments in most families of Caryophyllales. Unlike anthocyanins, betalains are derived from tyrosine via three enzymatic steps: hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA; step 1), and conversion of L-DOPA to betalamic acid (step 2), and to cyclo-DOPA (cDOPA; step 3). The principal enzymes responsible for these reactions have been elucidated at the molecular level, but their subcellular localizations have not been explored; hence, the intracellular compartments wherein betalains are biosynthesized remain unknown. Here, we report on the subcellular localization of these principal enzymes. Bioinformatic predictors and N- and C-terminal GFP tagging in transgenic tobacco, showed that Beta vulgaris CYP76AD1 which mediates both steps 1 and 3, DODA1 that catalyzes step 2, and CYP76AD6 which also mediates step 1, were similarly localized to the cytoplasm and nucleus (although the P450s were also weakly present in the endoplasmic reticulum). These two compartments were also the principal locations of Mirabilis jalapa cDOPA5GT. The cytoplasmic and nuclear co-localization of these key enzymes in tobacco suggests that betalains are biosynthesized in the cytoplasm and/or nucleus of betalain-containing plant cells. Elucidation of the subcellular compartmentation of betalain biosynthesis will facilitate the bioengineering of the betalain biosynthetic pathway in non-betalain-containing plants.
Highlights
Betalains are a group of water-soluble nitrogenous pigments, comprising yellow betaxanthins and red-violet betacyanins
Prior to the experimental work, we used a variety of online bioinformatics resources, both individual predictor and integrator programs, to predict signal peptides, especially the chloroplast transit peptide, and the subcellular localizations of CYP76AD1 (AD1), CYP76AD6 (AD6), DODA1, and cDOPA5GT (5GT)
None of the four proteins was predicted to be localized to the nucleus, though a very low probability existed in the case of 5GT, whereas all the integrated prediction programs, except for PredSL, forecast a cytoplasm or plasma membrane localization for DODA1 and AD1 (Table 2 and Supplementary Table S1)
Summary
Betalains are a group of water-soluble nitrogenous pigments, comprising yellow betaxanthins and red-violet betacyanins They occur in most families of the order Caryophyllales, where they replace anthocyanins as major plant pigments. Their biosynthetic pathway is very simple, and essentially involves only three principal enzymatic steps (Figure 1; Strack et al, 2003; Han et al, 2009; Hatlestad et al, 2012; Gandia-Herrero and Garcia-Carmona, 2013; Schwinn, 2016): (1) 3 -hydroxylation of L-tyrosine to form L-3,4dihydroxyphenylalanine (L-DOPA), (2) conversion of L-DOPA to betalamic acid (BA), the common chromophore of the yellow and red betalains, and (3) transformation of L-DOPA to cycloDOPA (cDOPA). This redundant role has been attributed to beet CYP76AD5 (Sunnadeniya et al, 2016) and proposed for the monophenolase activity of the polyphenol oxidase (PPO)-type tyrosinase (Mueller et al, 1996; Steiner et al, 1996; Harris et al, 2012), as well as for the catalase–phenol oxidase (CATPO; Teng et al, 2016) in mushrooms and higher plants
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